The molecular aspects of the earliest steps in embryonic stem (ES) cell differentiation remain poorly understood. Our preliminary data suggest that global histone acetylation may be a critical first step in differentiation. The goals of this proposal are to establish whether global histone acetylation and/or methytation occurs during multipie hormonally induced methods of ES cell differentiation and establish the time frame in which these histone modifications occur using standard assays for histone modifications. Our model predicts that the bulk of these histone modifications probably occur in promoter regions and we will make use of novel CpG island arrays to confirm this. Studies are designed to determine whether the global histone modifications that occur during exit from the undifferentiated ES cell state are uniquely different from the gene-specific histone modifications induced by hormonal signallingto highly differentiated cells. The second specific aim is to explore the possible mechanisms leading to these unique global histone modifications early in ES cell differentiation by screening ES cell extracts for various enzymatic activities responsible for covalent modification of histories. The third specific aim is designed to test the functional significance of histone modifications in directly regulating ES cell differentiation. The histone deacetylase inhibitor, trichostatin A (TSA), will be used in conjunction with specific growth factors to increase the rate and proportion of cells directed to a specific committed cell fate. In addition, overexpression of specific gene products designed to inhibit histone acetyltransferase activity or increase histone deacetylase activity in ES cells will be assessed for effects on the rate of ES cell differentiation and overall cell fate commitment. The proposed studies are designed to understand early hormonally regulated ES cell differentiation with potential application for significantly improving the yield of lineage-specific differentiation in vitro. This would greatly facilitate the development of ES cell technology for potential transplantation of "pure" cell populations into patients with diseases such as Parkinson disease, aplastic anemia, etc. In addition, the model proposed within this application predicts that histone deacetylase inhibitors such as valproic acid (a known teratogen) and TSA may greatly potentiate the teratogenic effects of environmental compounds by altering the very early histone acetylation pattern required for normal lineage-specific differentiation.